1. Field of the Invention
The present invention relates generally to waveguide-binding sensors for use in fluorescence assays, and, more particularly, to methods for increasing the sensitivity of a fiber-optic waveguide-binding sensor which remotely senses fluorescence radiation during assays of liquid solutions.
2. Background of the Invention
The evanescent wave portion of an electromagnetic field produced by light propagating through an optical waveguide characteristically penetrates a few hundred nanometers into the medium surrounding the optical waveguide. This evanescent wave can excite fluorescent molecules, e.g., fluorophores, to fluoresce when binding pairs of molecules are near the optical waveguide surface. The application of this phenomenon to an immunoassay sensor, wherein the biological recognition (binding) of an antigen by antibodies attached to the waveguide surface with concomitant diplacement of fluorescent-labeled antigen is measured as a change in fluorescence, was first disclosed in "A New Immunoassay Based on Fluorescence Excitation by Internal Reflection Spectroscopy" by Kronick and Little, Journal of Immunological Methods, 1975, Vol. 8, page 235.
The use of optical fibers as a special class of waveguides for immunoassay sensors is also known. For example, U.S. Pat. No. 4,447,546 discloses the use of optical fibers as waveguides which capture and conduct fluorescence radiation emitted by molecules near their surfaces. However, conventional waveguide-binding sensors for use with assays of aqueous fluids have demonstrated inadequate sensitivity. Specifically, poor sensor performance is attributed at least in part to the small size of the sample being analyzed, typically one or several monolayers in depth, and the small surface area of the optical waveguide, which factors limit the number of fluorophores which may be excited. More serious sensor performance degradation is mainly attributable to the effects of a weak evanescent wave, which fails to excite the fluorophores enough to produce detectable levels of fluorescence.
Increasing the strength of the evanescent wave penetrating into a fluid sample to be assayed increases the amount of fluorescence, thereby increasing sensor sensitivity. However, the evanescent wave propagates as higher-order modes within and near the outer surface of an optical waveguide core. These higher-order modes are weakly-guided, lossy, and can easily leak at a discontinuity or a bending point along the waveguide.
The use of tapered optical fibers to increase the sensitivity of fiber-optic assay systems is known. For example, U.S. Pat. Nos. 4,654,532 and 4,909,990 disclose the use of tapered fibers to increase the numerical aperture of the proximal end of the sensor, i.e., the sensing portion located at the near end of the fiber, as a means of increasing sensor sensitivity. In the disclosed systems, an unclad tapered optical fiber which is completely isolated from the sample fluid is mounted on the proximal end of a sensing fiber. The tapered portion forces the convergence of input excitation radiation prior to entry into the sensing portion of the fiber, thus increasing the numerical aperture from the proximal to distal end and, allegedly, increasing sensor sensitivity.
The present inventors recognize, however, that the introduction of a tapered section of the optical waveguide at the proximal end fails to address certain important issues central to the sensitivity of these sensors, especially in remote sensing applications. In particular, the higher order modes propagating in the section of the waveguide where the fluorophores are found (the distal end) comprise the evanescent wave and the bulk of the fluorescence coupled back into the fiber. These higher order modes typically propagate with greater loss than lower order modes.
The sensitivity of known fiber-optic sensors is further reduced by loss of poorly-guided fluorescence radiation propagating along the length of an optical fiber. Like the evanescent wave, fluorescence radiation from sources on the fiber core surface propagates in the higher-order modes and is equally susceptible to losses due to microbending of the optical fiber.
Known fiber-optic sensors suffer from the additional disadvantage that their performance is dependent on the length of the waveguide.